To dissect the mechanism of ubiquitin-dependent regulation of ribosome biogenesis and translation during neural crest and craniofacial development We had previously identified the multi-subunit ubiquitin E3 ligase CUL3 RING Ligase (CRL3) with its vertebrate-specific substrate adaptor KBTBD8 (CRL3KBTBD8) as an essential regulator of human and Xenopus tropicalis neural crest cell formation. We showed that CRL3KBTBD8 ubiquitylates the ribosome biogenesis regulator NOLC1 and its paralog TCOF1, whose mutation underlies Treacher Collins Syndrome, a neurocristopathy characterized by loss of cranial neural crest cells and defects in craniofacial development. Intriguingly, CRL3KBTBD8 controls neural crest cell formation and craniofacial development by regulating the function of newly synthesized ribosomes to alter the translational program of differentiating cells. However, the precise molecular mechanism of ribosome specification and the signaling networks that integrate this novel type of regulation into the differentiation program have remained elusive. During the last funding period, we have been able to show that CRL3KBTBD8-dependent monoubiquitylation and neural crest specification require multisite substrate phosphorylation by CK2, a kinase whose levels gradually increase during development of the nervous system. The essential CRL3KBTBD8-substrates TCOF1 and NOLC1 contain ten or more motifs that, following their phosphorylation by CK2, can be independently recognized by a conserved surface on KBTBD8. We found that seven or more CK2 motifs need to be present in the same substrate to mediate both monoubiquitylation by CRL3KBTBD8 as well as neural crest specification. Multisite dependency allows cells to convert a gradual increase in kinase inputs, as seen for embryonic CK2, into decisive activation of signaling outputs. We therefore propose that multisite dependency of CRL3KBTBD8 provides an elegant mechanism for switch-like cell-fate decisions controlled by monoubiquitylation. Taken together, our findings uncover an important role for multisite phosphorylation in integrating ubiquitin-dependent regulation of ribosome biogenesis and function into the neural crest differentiation program (Werner et al, 2018, Elife). Our study further identifies an essential role for dimerization for KBTBD8s function in cell-fate determination. In a collaborative study led by the laboratory of Michael Rape at UC Berkeley, we could show that KBTBD8 dimerization is subject to a quality control pathway that ensures proper neural crest formation (Mena et al, 2018, Science). To elucidate novel roles for specific CUL3-RING ubiquitin ligases in hESC maintenance and differentiation Amongst 600 human E3s, Cul3-RING Ligases (CRL3s) are a family of multi-subunit E3s that use 90 BTB domain-containing proteins as substrate adaptors. While particular CRL3-BTB complexes are known to regulate crucial aspects of human development and physiology, biological functions of the majority of the CRL3 E3s are still to be discovered. It is the major goal of this aim to identify novel roles for CRL3-BTB complexes in development and dissect the molecular underpinnings of their mechanism of action. We have previously identified a particular CUL3-BTB complex as an essential regulator of hESC actin dynamics and of neuronal differentiation from embryoid bodies. During the last funding period, through combining proteomic and biochemical approaches with hESC differentiation assays, we have identified an actin cytoskeleton signaling module through which the CUL3-BTB complex regulates embryoid-body based neural progenitor and neuron formation. Since mutations in many of the components of this signaling module are known to cause neurodevelopmental diseases, we hypothesized that also mutations in the CUL3-BTB complex itself could cause defects in human neurodevelopment. Thus, in collaboration with the lab of Daniel Kastners lab (NHGRI), we systematically queried exome databases for BTB variants in patients with undiagnosed developmental diseases, focusing on those with rare allele frequency and located in functional protein domains. Indeed, we found mutations in two phenotypically overlapping patients both exhibiting intellectually disability and structural brain malformations. Intriguingly, using immunoblotting and co-immunoprecipitation, we can show that these patient variants either reduce binding to the catalytic subunit CUL3 or to the actin cytoskeleton signaling module. This strongly suggests that ubiquitylation activity by the CUL3-BTB complex is required for signaling through the actin cytoskeleton signaling module for proper neuronal development and, if reduced, leads to neurodevelopmental disease. Our current efforts are geared towards mechanistically dissecting these processes. To dissect the functions and mechanism of deubiquitylases during embryonic development Ubiquitylation, the covalent attachment of ubiquitin to proteins, is an essential post-translational modification that orchestrates many aspects of human development. Through attachment of either one ubiquitin molecule or chains of ubiquitin typically linked through different K residues, ubiquitylation is able to regulate various substrate fates ranging from substrate degradation to control of intracellular signaling pathways. Deubiquitylases of the ovarian tumor family (OTU DUBs) are important regulators of the ubiquitin code and control crucial aspects of human physiology. OTU DUBs elicit their functions by targeting distinct linkage types within polyubiquitin to modulate the stability, activity, or interaction landscapes of their substrates. While some OTU DUBs are well characterized and have been linked to monogenetic diseases, the physiological functions and underlying mechanisms of the majority of OTU DUBs have remained largely elusive. During the last funding period, we have identified a cohort of patients with missense and nonsense mutations in several OTU DUBs all with developmental delay and multiple congenital anomalies or neurodevelopmental defects. Thus, we hypothesize that linkage-specific deubiquitylation activity of OTU DUBs is essential to edit ubiquitin modifications to ensure proper ubiquitin signaling during embryonic development. It is the major goal of this aim to mechanistically dissect how defects in activity of particular OTU DUBs lead to developmental disease. We are currently combining hESC/iPSC culture with biochemical and proteomic approaches to dissect the mechanisms of how mutations in these DUB genes lead to these severe phenotypes.